1. Field of the Invention
The present invention relates to a microfluidic system for assembling and for subsequently analysing complex cell arrangements, and to a corresponding method.
2. Related Prior Art
In many areas of scientific research and of diagnosis, whether in a research laboratory or in the daily work of a laboratory concerned with routine investigations, there is a need for complex cell arrangements which can be perfused under conditions which are as physiological as possible, meaning for example being present in the anatomically correct arrangement of the individual cell types relative to one another and/or physiologically functionally.
One example of the use of such complex cell arrangements is the determination of the toxicity and metabolism of medicaments in the pharmaceutical industry.
At present, the toxicity of medicaments is determined using 2D cell cultures in vitro, but the predictive power thereof for the effect of the medicaments in vivo is only low. One reason for this is the fact that the currently available complex cell arrangements which can be used for corresponding investigations in vitro do not, owing to their structure and arrangement, exhibit the same properties as corresponding cell or tissue structures in vivo.
The result thereof is an only limited informative power of the experiments carried out with the known cell cultures in relation to behaviour (toxicity, metabolism, mechanisms of action) in vivo, so that for example side effects of medicaments are often discovered only in clinical studies when the product is administered to patients, and large expenditures have already been made for research and development.
Another approach to the determination of the toxicity of medicaments are animal experiments, but use thereof is declining, besides their informative power being only provisionally applicable to humans, also for ethical reasons.
A further approach consists of investigating the effect of medicaments on isolated cells, but the predictive power is also limited here, as with the abovementioned 2D cell cultures, since single cells or two-dimensional cell arrangements differ in essential with functions from those of the three-dimensional “natural” cell assemblage.
Hence there is a need for complex, organotypical cell culture systems consisting of “natural” cells which grow in environments which allow differentiation over an appropriately long period of time, and a function comparable to the in vivo situation.
Of particular interest in this connection is on the one hand an organotypical liver cell co-culture with which medicaments are to be tested for toxicity and metabolism. The liver serves inter alia for the degradation and excretion of metabolic products, medicaments and toxins which enter the liver via the circulatory system. These substances are metabolized by the hepatocytes and transported out with the bile fluid. The bile fluid produced by the liver enters the intestine via the biliary tract and is excreted in this way. It is important for an organotypical liver cell culture for medicament testing that the hepatocytes are, to the outside, invested by endothelial cells, and perfusion of the complex cell culture takes place from the side of the endothelial cells. The co-culture of hepatocytes with endothelial cells and, where appropriate, stellate cells ensures the tissue-typical differentiation of the hepatocytes and, associated therewith, expression of genes necessary for metabolizing the substances mentioned.
There is also a need for an organotypical tissue structure like that to be found for example in the intestine. In this case, too, it is necessary to distinguish between “inside” and “outside” for physiologically functional perfusion. Consumed substances are enzymatically cleaved in the intestine and transported into the blood stream via the intestinal epithelium. The intestinal epithelium consists of a monolayer epithelial layer facing the intestinal lumen, and an underlying layer of mesenchymal cells which maintains the differentiation and function of the epithelial cells. It would be possible to carry out investigations on the uptake of medicaments on oral administration in such a cell assemblage produced in vitro.
A further area of use is the so-called blood-brain barrier which controls the penetration of substances from the blood into the brain and ensures that the chemical composition of the intracellular fluids of the brain remains substantially constant, as is necessary for precise signal transmission between the nerve cells of the central nervous system. The blood-brain barrier is formed by endothelial cells and astrocytes around blood vessels. They ensure, via active transport systems, the transfer of nutrients and oxygen or metabolites. Knowledge about the permeability of the blood-brain barrier for active ingredients and thus their availability in areas of the nervous system is of particular interest in connection with the development of active ingredients.
The publication “Rapid Heterogenous Liver-Cell On-Chip Patterning via the Enhanced Field-induced Dielectrophoresis Trap” by Ho, et al., Lab Chip, 2006, 6, 724-734, discloses a microfluidic chip on which it is possible to establish a planar structure of liver cells, i.e., a 2D arrangement. An inhomogeneous electric field with defined gradients is generated through the geometric structure and arrangement of the electrodes and brings together cells of two cell types which are randomly present in a chamber to give a desired planar tissue pattern.
The authors mention that microfluidic patterning with microchannels and laminar flow cannot be applied to liver cells because this method is too coarsely structured in its effect. In addition, positive dielectrophoresis is described as a possible way of actively manipulating cells. However, the authors mention that this method has not yet been successfully employed to assemble complex cell arrangements.
With this background, the authors propose employing microfluidics together with a specially structured electric field in order to produce desired tissue patterns. For this purpose, the chip comprises a cell structuring chamber fed via microchannels continuously with cells which associate to give the complex structure via the electric field formed in the chamber. The electric field is then switched off, and pure medium is pumped through or into the chip.
The cell arrangement which can be generated with the known apparatus is, however, planar, so that the disadvantages described above arise for use in pharmacological research. It is not possible with the apparatus and the method of Ho, et al., to generate a complex cell arrangement which can be physiologically functionally perfused and can be employed as organotypical tissue for example for toxicity measurements.